Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants
| Autor: | Inacrist Geronimo, W.E. Rogers, Christina M. Payne, T. Othman, David K. Heidary, Edith C. Glazer, Catherine A. Denning, Tom Huxford |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Models Molecular Circular dichroism Cytochrome Stereochemistry Protein Conformation Palmitic Acid Crystallography X-Ray Hydroxylation 01 natural sciences Biochemistry Article Electron Transport 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Cytochrome P-450 Enzyme System Oxidoreductase Enzyme Inhibitors Heme Bacillus megaterium NADPH-Ferrihemoprotein Reductase chemistry.chemical_classification Binding Sites biology 010405 organic chemistry Chemistry Circular Dichroism Substrate (chemistry) Metyrapone biology.organism_classification Protein tertiary structure 0104 chemical sciences Kinetics 030104 developmental biology Mutation biology.protein Mutagenesis Site-Directed Mutant Proteins Salt bridge Crystallization Oxidation-Reduction |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 |
| Popis: | Cytochrome P450(BM3) is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450(BM3) mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450(BM3) variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability. |
| Databáze: | OpenAIRE |
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